**2. Preclinical trials of technologically processed highly diluted antibodies to S100 protein**

#### **2.1 Pharmacodynamics**

#### *2.1.1 Biological activity*

#### *2.1.1.1 Antistress activity of TP Abs to S100*

Antistress activity of TP Abs to S100 was studied using three approaches.

#### *2.1.1.1.1 Effect on somato-vegetative manifestations of stress*

Negative emotions arising from stress caused by the anticipation of pain or other negative expectations (in particular, on the eve of surgical operations, educational tests, important meetings, etc.) are accompanied by anxiety and fear. Concurrently, a cascade of somato-vegetative manifestations of stress is initiated [61].

Modeling of a conditioned emotional reflex to unescapable electric pain stimulation was performed on outbred white male rats weighing 220–280 g [27]. This was followed by monitoring of animal behavior in a stressful situation (repeated placement in an experimental 'dangerous' camera) as well as emotional responses when stress was intensified by an additional negative provocation (approaching an unfamiliar object to the animal's head). Antistress activities of TP Abs to S100 and diazepam ('classical' benzodiazepine tranquilizer, positive control) were estimated by administering drugs one day after development of the conditioned reflex.

Rats in the control group (hereinafter, animals that received distilled water as a placebo) when they were subsequently placed in a "dangerous" chamber responded by freezing (45%) or actively trying to escape the chamber (35%) (**Table 1A**). Only 20% of rats showed calm behavior. At the same time, somato-vegetative manifestations of stress were observed in animals (especially with a passive reaction): increased frequency of breathing, urination, defecation, and squeaking. Both TP Abs to S100 and diazepam caused a decrease in the number of rats with a passive and active response to stress, as well as significantly (three times) increased the number of animals with a calm orientation-exploratory activity. Somato-vegetative manifestations of stress also dissipated in both groups.

The emotional reaction of anxiety and anxiety associated with the expectation of pain in a "dangerous" chamber was significantly enhanced when using additional provocation—bringing an unfamiliar object to the head of the animal. This was manifested as an increase in the number of rats with active (up to 40%) and passive (up to 55%) behavior and a decrease in the number of animals with calm behavior (down to 5%) (**Table 1B**). Respiratory symptoms, squeaking, frequency of defecation, and urination also increased. Both drugs (TP Abs to S100 and diazepam) reduced the severity of stress induced by expectation of pain. TP Abs to S100 reduced both the number of animals with a spontaneous active and passive reaction by 20%, while diazepam reduced the number of animals with active attempts to escape the chamber (by 35%) more than the number of animals with freezing (only by 10%). The same trend continued with additional negative provocation, which may be the result of the sedative activity of diazepam, which TP Abs to S100 do not have.


*Technologically Processed Highly Diluted Antibodies to S100 Protein in the Treatment… DOI: http://dx.doi.org/10.5772/intechopen.92207*

*Note: animals were intragastrically administered distilled water (2.5 ml/kg, control),TP Abs to S100 (2.5 ml/kg), or diazepam (1 mg/kg) at a single dose 30 minutes prior to testing. n/a, not applicable; TP Abs to S100, technologically processed highly diluted antibodies to S100 protein.*

*\** р *< 0.05 versus corresponding control.*

*Bold entries were made to emphasize the results in TP Abs to S100 group.*

#### **Table 1.**

*TP Abs to S100 antistress activity in a model of a conditioned emotional reflex to unescapable electric pain.*

#### *2.1.1.1.2 Effect on c-Fos protein expression*

It is known that immediate-early response *c-fo*s gene expression in the hypothalamic paraventricular nucleus is one of the primary biological markers of stress [62]. The effectiveness of stress-protective compounds can be assessed by their ability to suppress *c-fos* expression in the brain.

The study was conducted on male Wistar rats weighing 250–280 g [63], classified as active or passive (stress-resistant or predisposed to stress, respectively) in the open field (OF) test [64]. The OF test is widely used to study the behavior of rats [65]: animals are placed in the center of the OF arena and the horizontal and vertical activity, the number of entries into the center zone, as well as the number of acts of defecation and urination (emotionality) is recorded.

Rats were administered TP Abs to S100 or imipramine (antidepressant drug that modulates *c-fos* expression) and then subjected to 1-hour immobilization with simultaneous electrocutaneous irritation. Immunohistochemical detection of c-Fos protein in the parvocellular neurons of the paraventricular nucleus of the hypothalamus was performed in samples obtained 90 min after the procedure, at the peak of the protein expression [62].

In response to stress, c-Fos protein level significantly increased (vs. intact animals) in both active and passive animals (20–25 fold), and in the latter, this increase was more pronounced (**Figure 1**). TP Abs to S100 and imipramine demonstrated equally and pronounced antistress activity in passive animals: 1.2- and 1.5-fold decrease in the number of Fos-positive cells was observed, respectively.

#### **Figure 1.**

*TP Abs to S100 effect on c-Fos protein expression (stress marker) in the rat hypothalamic paraventricular nucleus after 1-hour immobilization with simultaneous electrocutaneous irritation. Note: animals were intragastrically administered distilled water (2.5 ml/kg, control),TP Abs to S100 (2.5 ml/kg) or imipramine (12 mg/kg) at a single dose or for 20 days preceding stress exposure. Data are expressed as M SD. \**р *< 0.05 (#* р *< 0.001) versus corresponding control. TP Abs to S100, technologically processed highly diluted antibodies to S100 protein.*

#### *2.1.1.1.3 Effect on gastric ulcers after an immobilization stress*

Another important biological marker of stress is development of ulcers in the gastric mucosa. For example, it is known that immobilization stress is accompanied by severe gastric ulceration [66].

The study of antistress activity of TP Abs to S100 was carried out on male Wistar rats weighing 250–280 g, classified as active or passive in the OF test [67]. Animals were administered TP Abs to S100 at a dose of 2.5 ml/kg for 5 consecutive days or placebo. On the 6th day, half of the rats from each group were immobilized by fixing their paws on a special platform for 1 h, and then the number of animals with ulcers and total number of ulcers formed in the stomach was counted.

TP Abs to S100 decreased by 33.4% the number of animals with ulcers in the group of passive (but not active) rats, which complements the previously obtained results on the higher efficacy of the drug in passive, highly sensitive to stress animals.

TP Abs to S100 also reduced the total number of ulcers in both groups by more than 50%. Again, in control passive animals, there were 1.3 times more ulcers than in control active ones. However, after TP Abs to S100 administration, there was no such difference.

#### *2.1.1.2 Anxiolytic activity of TP Abs to S100*

The studies were carried out on outbred white male rats weighing 230–250 g [31] using the most widely validated tests (the Vogel conflict test, the elevated plus maze test, and the OF test) [65]. The activity of TP Abs to S100 was compared to diazepam.

The conflict situation in the *Vogel test* was created by exposing animals to opposing behavioral tendencies: motivation to drink and fear, when every attempt to drink was punished by an electric shock. This lead to a significant reduction in water consumption. Drugs with anxiolytic properties alter behavior and cause an increase in drinking.

#### *Technologically Processed Highly Diluted Antibodies to S100 Protein in the Treatment… DOI: http://dx.doi.org/10.5772/intechopen.92207*

To study the activity of TP Abs to S100, depending on the individual reaction to stress, animals were grouped into highly (stress-resistant) and low active (predisposed to stress) in the forced swim test with water wheel (Nomura test), in which stress is modeled, and asthenia and depressive behavior are evaluated. Then, animals were treated with TP Abs to S100 or diazepam, and the Vogel conflict test was performed.

Anxiolytic effect of TP Abs to S100 was not inferior to that of diazepam: the number of punished water intakes in highly active groups increased by 27.4 and 28.7%, respectively (**Figure 2**). Meanwhile, in low-activity animals characterized by a predisposition to asthenia and depressive behavior [64], TP Abs to S100 efficacy was superior to diazepam (2.8 and 2 times vs. control, respectively). The data obtained indicate that in addition to the anxiolytic activity TP Abs to S100 have an antiasthenia activating effect, which distinguishes them from diazepam that induces both anxiolytic and sedative effects.

The *elevated plus maze test* is based on the fear of heights and open spaces: animals are placed on the central platform of the maze and the latent period before the first entry into the open arms, the number of full and incomplete entries and the duration of stay in them, as well as the number of head dips below the level of the open arms is recorded.

It was established that TP Abs to S100 and diazepam had a similar anxiolytic effect in this test: both drugs increased the number of entries into the open arms (1.9 and 2.3 times, respectively), the time spent in the open arms (5.4 and 7 times), as well as the number of head dips (5 and 9 times) versus control animals (**Table 2**).

In the *OF test*, the antianxiety activity of TP Abs to S100 and diazepam was demonstrated by the fact that rats began to go to the center of the field, which was not observed in the control group (**Table 3**). However, unlike diazepam, which reduced the horizontal activity of animals by 1.5 times, TP Abs to S100 did not change this parameter and, therefore, did not have a sedative effect.

#### **Figure 2.**

*TP Abs to S100 demonstrate anxiolytic activity in the Vogel conflict test. Note: animals were intragastrically administered distilled water (2.5 ml/kg, control),TP Abs to S100 (2.5 ml/kg) or diazepam (2 mg/kg) at a single dose 30 minutes prior to testing. Data are expressed as M SD. \**р *< 0.05 versus corresponding control. TP Abs to S100, technologically processed highly diluted antibodies to S100 protein.*


*Note: animals were intragastrically administered distilled water (2.5 ml/kg, control),TP Abs to S100 (2.5 ml/kg), or diazepam (2 mg/kg) at a single dose 30 minutes prior to testing. Data are expressed as M SD. TP Abs to S100, technologically processed highly diluted antibodies to S100 protein.*

*\** р *< 0.05 versus control.*

*Bold entries were made to emphasize the results in TP Abs to S100 group.*

#### **Table 2.**

*TP Abs to S100 anxiolytic activity in the elevated plus maze test.*


*Note: animals were intragastrically administered distilled water (2.5 ml/kg, control),TP Abs to S100 (2.5 ml/kg), or diazepam (2 mg/kg) at a single dose 30 minutes prior to testing. Data are expressed as M SD. TP Abs to S100, technologically processed highly diluted antibodies to S100 protein.*

*\** р *< 0.05 versus control.*

*Bold entries were made to emphasize the results in TP Abs to S100 group.*

#### **Table 3.**

*TP Abs to S100 anxiolytic activity in the open field test.*

#### *2.1.1.3 Antiaggressive activity of TP Abs to S100*

Anxiety disorders are often accompanied by covert or overt aggression. The antiaggressive activity of TP Abs to S100 was studied in the tests of motivated and unmotivated aggression on outbred adult white male rats weighing 200–250 g in comparison with diazepam [68].

In the *test of unmotivated aggression caused by inescapable shock*, the threshold of aggressive response of a pair of animals placed on a grid floor was determined by increasing the stimulating current. Animals manifested shock-elicited aggression when they assumed upright "boxing" posture and tried to bite and strike each other with front and hind paws.

TP Abs to S100 and diazepam after a single dose and course administration exerted antiaggressive activity: single TP Abs to S100 administration increased the threshold of aggressive response by 23.1%, and after a 4-day administration—by 31.3% compared with the control, while diazepam increased this threshold by 26.3 and 34.9%, respectively (**Figure 3**).

The *test of motivated aggression* is based on the study of the intensity of the aggressive reaction elicited in a pair of rats trying to escape electric shock. Rats were individually taught to avoid pain caused by electric irritation of the paws on a safe bench installed in the center of the chamber. Then, they were placed in pairs in the chamber, and their behavior was observed for 2 min. Control animals began to fight for a safety on the bench, which had a capacity to tightly fit both animals. The criterion for the effectiveness of substances with antiaggressive action in this test was the duration of joint avoidance of pain exposure.
